RESUMEN
Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1ß than PfklS775A/S775A after LPS treatment. In an in vivo inflammation model, PfklS775A/S775A mice show reduced levels of MCP-1 and IL-1ß. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis.
Asunto(s)
Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Inmunidad Innata , Interleucina-1beta , Macrófagos , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Ratones , Fosforilación , Interleucina-1beta/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Reconocimiento de Patrones/genética , Fosfofructoquinasa-1/metabolismo , Fosfofructoquinasa-1/genética , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Humanos , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Inflamación/metabolismo , Masculino , Reprogramación MetabólicaRESUMEN
S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel KV1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Gasderminas , Neutrófilos/metabolismo , Selectina E/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Inflamación/metabolismoRESUMEN
The release of neutrophils from the bone marrow into the blood circulation is essential for neutrophil homeostasis and the protection of the organism from invading microorganisms. Granulocyte colony-stimulating factor (G-CSF) plays a pivotal role in this process and guides granulopoiesis as well as the release of bone marrow neutrophils into the blood stream both during homeostasis and in case of infection through activation of the G-CSF receptor/signal transduction and activation of transcription 3 (STAT3) signaling pathway. Here, we investigated the role of the mammalian sterile 20-like kinase 1 (MST1) for neutrophil homeostasis and neutrophil mobilization. We found increased plasma levels of G-CSF in Mst1 -/- mice compared to wild type mice both under homeostatic conditions as well as after stimulation with the proinflammatory cytokine TNF-α. In addition, G-CSF-induced mobilization of neutrophils from the bone marrow into the blood circulation in vivo was markedly reduced in the absence of MST1. Interestingly, this was not accompanied by differences in the number of blood neutrophils. Addressing the underlying molecular mechanism of MST1-regulated neutrophil mobilization, we found reduced STAT3 phosphorylation and impaired upregulation of CXCR2 in Mst1 -/- bone marrow neutrophils compared to wild type cells, while JAK2 phosphorylation was not altered. Taken together, we identify MST1 as a critical modulator of neutrophil homeostasis and neutrophil mobilization from the bone marrow, which adds another important aspect to the complex role of MST1 in regulating innate immunity.
Asunto(s)
Médula Ósea , Neutrófilos , Receptores de Factor Estimulante de Colonias de Granulocito , Factor de Transcripción STAT3 , Animales , Ratones , Factor Estimulante de Colonias de Granulocitos/metabolismo , Homeostasis , Transducción de Señal , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
AIMS: Neutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling. METHODS AND RESULTS: Using in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3. CONCLUSION: We show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.
Asunto(s)
Canales de Potasio con Entrada de Voltaje , Animales , Calcio/metabolismo , Inflamación , Canal de Potasio Kv1.5 , Potenciales de la Membrana/fisiología , Ratones , Infiltración NeutrófilaRESUMEN
Recruitment of neutrophils from the intravascular compartment into injured tissue is an essential component of the inflammatory response. It involves intracellular trafficking of vesicles within neutrophils and endothelial cells, both containing numerous proteins that have to be distributed in a tightly controlled and precise spatiotemporal fashion during the recruitment process. Rab proteins, a family of small GTPases, together with their effectors, are the key players in guiding and regulating the intracellular vesicle trafficking machinery during neutrophil recruitment. This review will provide a short overview on this process and highlight new findings as well as current controversies in the field.